B-cell chronic lymphocytic leukemia (B-CLL), represents the most common leukemia in Western countries and has an estimated incidence of 1 per 100,000 per year. It is characterized by the monoclonal expansion of B lymphocytes expressing the CD5 marker and exhibiting a long life span, possibly because of a perturbed apoptotic program.
Current knowledge of the molecular pathogenesis of B-CLL is limited because no specific genetic alteration has yet been associated with this disease. In particular, B-CLL is not associated with reciprocal balanced chromosomal translocations. Accordingly, none of the proto-oncogenes involved in chromosomal translocations in other mature B cell malignancies, including cyclin D1, BCL-2, BCL-6, PAX-5, and c-MYC, are primarily altered in B-CLL. Although in a fraction of B-CLL cases inactivation of the tumor suppressor gene p53 (on chromosome 17p13) and deletions or mutations of the ATM gene (on chromosome 11q22-23) have been reported, such lesions were observed in late stages of the disease and may not represent primary tumorigenic events. However, it is known that at the chromosomal level the 13q14 region of human chromosome 13 is very frequently deleted in several hematological malignancies. Chromosome 13q14 deletions (approximately 50% of the patients) are the most common chromosomal alterations associated with B-CLL (Dohner et al, J Mol Med.;77:266-281. 1999; Kalachikov et al, Genomics.;42:369-377. 1997), followed by structural aberrations of chromosome 11q (19% of the cases) and trisomy of chromosome 12 (15%). Moreover, chromosome 13 deletions can be the only cytogenetically detectable abnormality, suggesting an early role in B-CLL pathogenesis. Similarly, 13q14 alterations, mainly deletions, are frequently observed in acute leukemia, multiple myeloma and mantle cell lymphoma. Alterations of 13q14 (25-40% of samples) provide myeloma patients with a statistically significant adverse prognostic as an independent factor of outcome (Zojer et al, Blood 2000 95:1925-30; Shaughnessy et al, Blood 2000 96:1505-11). It has also been shown that deletion of chromosome 13 is associated with transition from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma. Deletion of 13q14 has been also observed in 50% of mantle cell lymphoma cases (Cuneo et al, Blood 1999 93:1372-80).
The karyotyping of CLL samples identified relatively few chromosomal abnormalities, suggesting that the specificity and frequency of observed deletions at 13q14 have pathologic significance. Several groups have used positional cloning to identify the gene or genes targeted by the deletions. A region of more than 1 Mb has been fully sequenced and characterized in detail (Bullrich et al, Cancer Res 2001 61:6640-8; Migliazza et al, Blood 2001 97:2098-2104). So far, a total of eight genes have been identified and screened for alterations at the DNA and/or RNA level in sporadic and familial cases of CLL: Leu-1 (BCMS or EST70/Leu-1), Leu 2 (ALT1 or 1B4/Leu-2), Leu 5 (CAR), CLLD6, KPNA3, CLLD7, LOC51131 (putative zinc finger protein NY-REN-34 antigen) and CLLD8. However, detailed genetic analysis, including extensive loss of heterozygosity (LOH), mutation, and expression studies, have failed to demonstrate the consistent involvement of any of the genes located in the deleted region.
With the definition of a minimally deleted region in 13q14.3 between markers D13S31 and RB1 it became possible to analyze larger numbers of tumors for loss of heterozygosity (LOH) status where it was soon shown that homozygous deletions were largely centered around the D13S25 locus (Chapman et al, Oncogene 1994 9:1289-93). The observation of homozygous deletions also demonstrated that it is the loss of function of a critical gene in 13q14 which is important in the development of B-CLL. Subsequent analyses of larger numbers of tumors confirmed the observation of homozygous deletions and enabled a refinement of the limits of the minimally deleted region (Bullrich et al, Blood 1996, 88: 3109-15; Bouyge-Moreau et al, Genomics 1997, 46:183-90).
Although there is some variation between reports attempting to define ever decreasing sizes of the critical region, these often depended on the observation in single tumors which might have been due to peculiar rearrangements in these cases, possibly unrelated to leukemogenesis. At present, the critical region is understood to be in a region approximately 700 kb and bounded by markers D13S319 and D13S25.
Accordingly, large screenings were undertaken using different approaches, of which FISH was one of the most prominent, using genomic probes specific for the markers RB1, D13S25 and D13S319 (commercially available now from companies like Vysis, Inc.). However, none of the genes and/or probes used in these screenings achieved a 100% detection rate of 13q14 deletions in CLL. The same region/genes of 13q14 were studied in multiple myeloma and mantle cell lymphoma but no consistent observations have been made for a potential tumor suppressor gene located at 13q14.
Subsequently, a project for the characterization of 13q breakpoints in acute leukemia was initiated which utilized a cell line from one of the patient samples harboring a unique t(12;13)(p12;q14) translocation (Meyer et al, Leukemia 2001 15(9): 1471-4). This cell line has been used to identify the gene(s) involved in this rearrangement to establish a molecular characterization of the pathogenetic events potentially leading to leukemia. One of the pathologic consequences of such a translocation is the creation of tumor-specific fusion genes from the juxtaposition of segments of DNA normally found on separate chromosomes. Recurrent chromosomal deletions, on the other hand, suggest the presence of a tumor suppressor gene within the deleted region, deletion of one allele being associated with mutation of the other. While the gene involved in this single translocation on chromosome 12p12 was identified as the ETV6 gene which has been involved in many balanced translocations, attempts to clone a potential partner gene were unsuccessful.
Thus, there is a need for a method of detecting chromosomal alterations in the 13q14 region of human chromosome 13 which indicate an individual is likely to have acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL).